Until now, substances suitable as imaging contrast agents in injectable compositions for blood-pool investigations have been mostly NMR responsive solid mineral and organic particles or water-soluble polymers. The particles can comprise ferromagnetic or superparamagnetic materials as well as paramagnetic species bonded to polymeric carriers. In order to make them sufficiently long lasting for imaging the circulation, the particles should be protected against premature removal from the bloodstream.
Normally, the useful life of particles injected in the circulation is short because of rapid physiological removal therefrom due to opsonization followed by phagocytosis. The opsonization process involves the coating of the particles by an antigen protein, opsonin, recognisable by macrophages. Then, in a second stage, opsonization is followed, by the phagocytosis and metabolization of the coated (opsonized) particles by the Kupffer cells of the liver and spleen. Hence, although unprotected particles are suitable for imaging of the liver and the spleen, their free life in the blood is too short for blood-pool imaging.
The protection of particles against early removal from the circulation is discussed in many documents and significant enhancement of their useful life in the blood has been achieved by coating magnetite particles with a coating which includes amphiphilic substances, for example ethyleneoxide-propyleneoxide block copolymers (see for instance WO-A-94/04197, Sintetica).
The use of dispersions of microvesicles containing concentrated solutions of iodinated or paramagnetic species encapsulated in the vesicles e.g. liposomes as carriers of X-ray opacifiers or NMR contrast agents has been proposed. Thus, EP-A-0 314 764 (Dibra) discloses injectable aqueous suspensions of liposomal vesicles carrying encapsulated at least one iodinated organic compound opaque to X-rays which are useful as contrast agents for X-ray imaging of liver and spleen. The liposomes have a mean size between 0.15 and 3 .mu.m and the ratio of the weight of the iodine encapsulated in the liposomes to the weight of the liposome forming lipids (I/L) is from 1.5 to 6 g/g. The liposome suspensions as carriers of opacifying compounds have been proposed due to their relative biocompatibility and ease of preparation.
Proposals to incorporate opacifiying agents into the liposome membranes have also been made (E. Unger et al., Liposome bearing membrane-bound complexes of manganese as magnetic resonance contrast agents, Proceedings of the Contrast Media Research Symposium, San Antonio Tex. Oct. 3-8, 1993, S168). However, most liposomes are subject to rapid removal from the circulation by the liver and the spleen and, although this property may be advantageous for imaging of these organs it is undesirable when blood pool imaging is contemplated. This since for blood pool imaging the concentrations of opacifying compounds in the blood should be kept at a relatively high level for extended periods of time.
To prolong the life of liposomes vesicles in the blood, different remedies have been proposed. Coating liposomes with copolymers containing hydrophilic and hydrophobic segments has been proposed in, for instance, J. Pharmacy & Pharmacol. 39 (1987), 52P, while incorporation of protective substances in the vesicle forming lipids has been proposed in EP-A-0 354 855 (Terumo) and in WO-A-91/05545 (Liposome Technology). Along the latter line of approach, "stealth factors", for instance, covalently modified lipids, i.e. lipids carrying grafted thereon externally extending polyethylene glycol (PEG) or polyoxyethylene-polyoxypropylene segments. Also, the incorporation, as "stealth" factors, to the vesicle forming lipids of products such as palmitoylglucuronic acid (PGlcUA) has been reported to improve the half-life of liposomes in the blood (see Naoto Oku et al. in Biochimica et Biophysica Acta 1126 (1992), 255-260).
EP-A-0 354 855 (Terumo) discloses use of agents for inhibiting adsorption of protein on the liposome surface comprising a hydrophobic moiety at one end and a hydrophilic macromolecular chain moiety on the other end. The preferred hydrophobic moieties are alcoholic radicals of long chain aliphatic alcohol, a sterol, a polyoxypropylene alkyl or a glycerin fatty acid ester and phospholipids while prefered hydrophilic moieties are polyethylene glycols (PEG). Non-ionic surface active agents in which PEG and an alcoholic radical of the hydrophobic moiety are bound by ether bond or PEG-bound phospholipids are particularly preferred. Upon formation the agent is admixed with liposome forming phospholipids to produce "stealth" liposomes.
The lifetime of liposomes in the blood may be significantly prolonged by making the vesicles very small, i.e. making them less size-recognisable by opsonin; this approach has been disclosed in WO-A-88/04924 and EP-A-0 442 962.
WO-A-88/04924 discloses liposome compositions containing an entrapped pharmaceutical agent in which the liposomes are predominantly between 0.07 and 0.5 .mu.m in size, have at least 50% mole of membrane-rigidifying lipid such as sphingomyelin or neutral phospholipids and between 5-20% mole of ganglioside GM.sub.1, saturated phosphatidyl inositol or galactocerebroside sulfate ester. From the disclosure (Examples 8 and 9) it follows that liposomes made with negatively charged phospholipids in which phosphatidyl moiety is linked to glycerol are not very useful for blood pool applications as the same are relatively quickly recognised by RES.
In EP-A-0 442 962 liposomes of 50 nm or less are proposed for transporting through the circulation minute amounts of drugs to selected areas in the body. The trouble with very small vesicles is that their entrapment capacity becomes very low and such small vesicles are not readily compatible with the amounts of contrast media required for imaging the blood-pool with paramagnetic or X-ray compounds. Thus, under the conditions disclosed it would be necessary to inject to live subjects liposome suspensions containing more than 100 mg of lipids/ml which is undesirable for reasons of cost, potential toxicity and very high viscosity. The use of tiny liposome vesicles of the kind proposed in EP-A-0 442 962 for the delivery of drugs (in the order of 50 nm or less) are therefore unpractical for blood-pool imaging. Much the same applies to the proposals of Gabizon et al. in Biochim. et Biophys. Acta 1103 (1992) 94-100 and I. A. J. M. Bakker-Woudenberg et al. ibid. 318-326 directed to liposomes with an average size between 0.07 .mu.m and 0.1 .mu.m and prolonged residence times in the blood.
From the recent publications of M. C. Woodle et al., Journal of Drug Targeting 2 (1994) 397-403 and I. A. J. M. Bakker-Woudenberg et al., ibid. 363-371, it follows that in view of a relatively rapid removal of even those very small liposomes, the presence of the recognised stealth factors is absolutely necessary if these liposomes are to be effective in transporting various targeted drugs. This then presents further problems as the production of liposomes with the "stealth factors" is rather cumbersome. In addition, the "stealth factored" liposomes are known to have very low entrapment capacity and while such liposomes may be suitable to carry specific drugs, and therefore useful in therapy, they are almost useless in imaging.
Hence, the problem of use of standard or unmodified liposomes i.e. liposomes which can carry sufficient amount of opacifier and remain in the blood circulation sufficently long to enable X-ray and NMR imaging remains unresolved. It is generally believed that, in addition to be able to supply sufficent amount of opacifier, for good blood pool imaging the contrast agent should upon administration remain in the circulation between 1 and 2 hours. Thereafter the blood pool contrast agent should be eliminated from the body as quickly as possible. Use of liposomes which would satisfy these criteria is desirable as liposome production techniques are well known; their use in medicine and diagnostic preparations is wide spread; their effects in the living body are reasonably well understood. Hence their use for blood pool imaging or the manufacture of blood pool agents of "stealth" liposomes without "stealth factors" would provide a number of advantages.
Actually, this problem has been unexpectedly solved by the present inventors according to the disclosure hereafter.